The integration of tools for manipulating and controlling cells within microfluidic systems has steadily grown due to various unique features that microscale technologies can provide in terms of fine control over cellular microenvironments, flow conditions, and precise cell positioning for specific cell-cell interactions. The combination of such tools with microsystems has enabled the study of cellular processes that otherwise would not have been possible. Among the tools currently available to position cells in precise locations on a substrate is dielectrophoresis (DEP), which is an electrokinetic technique that can trap particles (e.g. cells) based on polarizability differences between the particle and the media in which the particles are suspended when both are exposed to a non-uniform field. The use of DEP has been primarily limited to, short-term manipulation studies of cells or preparative methods to separate cells from complex mixtures. Few studies have demonstrated DEP trapping for long term cell experiments where cell function still remains days after the trapping is effected. Therefore, it is of paramount importance, when developing DEP devices for in vitro cell studies, to demonstrate that cell viability and cell function (e.g., proliferation, motility, differentiation) are maintained after the electrokinetic manipulation.
A typical design for using DEP to trap cells is the placement of DEP electrodes under a fluid flow in a microfluidic device. This arrangement allows for increased trapping of cells in a short time and the removal of untrapped cells from non-DEP parts of a substrate surface. A challenge to this design is retaining the trapped cells in a fluid flow field at the selected positions when the DEP forces are removed. In order to produce DEP forces capable of moving cells up the field gradient, known as positive DEP (pDEP), cells must be suspended in sucrose or other low conductivity media. As opposed to cells suspended in high conductivity media (e.g. cell growth media), pDEP conditions produce stronger traps, thus attracting more cells and holding them on the substrate while the DEP forces remain active. The difficulty with this arrangement occurs when the DEP forces are switched off and the fluid flow field dislodges the positioned cells. In order to maintain the cells in position one needs to have good control over flow so that cells may attach through their integrins or other adhesive proteins over a period of time. An alternative to controlling the flow by pumps and valve systems is to have a “sticky” surface to which cells will anchor to, immediately after DEP trapping is achieved. By taking advantage of the extracellular molecules around the cells, such as antibodies or glycoproteins, either specific or non-specific binding can be effected. In turn, this can produce cell attachment on the pretreated surface via antibody/antigen binding or electrostatic interactions. The latter approach has been investigated using polyelectrolyte multiple layers (PEMs) as the surface coating material and has been shown to work when anchoring cells for short term experiments. However, a more relevant material is needed for in vitro long term cell experiments not only to facilitate cell anchoring, but also to maintain cell proliferation and cell function.
The present inventors have demonstrated cell patterning using PEMs when seeded in cell culture medium as well as when trapped under DEP conditions. Cells trapped under DEP and PEM conditions showed that over 93% of the cells remained anchored on the PEMs after the electrodes were de-energized. However, further research has been conducted in an attempt to extend the use of this approach for long term cell experiments. The results obtained using PEMs and DEP conditions show deleterious effects on the cells 24 hours after DEP cell trapping (see FIGS. 1A and 1B). Specifically, referring to FIGS. 1A and 1B, P19 cells attached onto PEMs are shown under different conditions. FIG. 1A illustrates P19 cells 24 hours after DEP trapping in a microfluidic channel. Cells were anchored on PEMs while in sucrose media, and then the sucrose was replaced with cell growth media. The faint vertical lines in the center are the indium tin oxide (ITO) electrodes used for DEP trapping. FIG. 1B illustrates P19 cells 24 hours after seeded on PEMs in cell growth media. Note the difference between P19 cells poorly attached in FIG. 1A versus well-attached healthy cells shown in FIG. 1B. The scale bar in FIG. 1A is 100 μm. Therefore, an alternative to this approach is needed to achieve long term cell experiments using a “sticky” surface and DEP.
The precise positioning of cells is a key requirement when utilizing microfluidic systems, specifically when cells are needed to be in defined areas for their stimulation and study. A number of approaches have been introduced to manipulate or capture cells within microchannels. These approaches vary from mechanical traps and flow control, to optical and electronic techniques. Among the electronic techniques, dielectrophoresis (DEP) has gained much attention in the microfluidics community. This phenomenon was first described by Pohl in 1951. However, it was not until the last decade that the number of publications increased significantly for applications including biosensors, medical diagnostics, particle filtration, nanoassembly, and DNA manipulation. The main advantages that DEP offers for particle manipulation include label free entrapment, simplicity of instrumentation, favorable scaling effects, the ability to apply repulsive (negative DEP) and attractive (positive DEP) forces, and the lack of microfabricated obstacles that distort the flow within the channels. DEP coupled with lab-on-a-chip devices have demonstrated suitability for DEP-based cell applications such as separation by size, sorting, focusing, filtration, trapping, and patterning.
In general, DEP electrodes have been patterned on solid substrates such as glass slides and silicon wafers. However, few previous efforts have investigated patterning electrodes, for purposes other than DEP, onto permeable surfaces. For example, Duan and Meyerhoff showed that metallization of permeable membranes was possible and used patterned nylon membranes for sandwich enzyme immunoassays. Later, {hacek over (S)}vor{hacek over (c)}ik et al. characterized the sputtering process to metallize polyethylene terephthalate (PET).